The present invention concerns an antibiotic substance of microbial origin, arbitrarily denominated GE23077 complex and the individual factors that constitute it, namely GE23077 factor A1, GE23077 factor A2, GE23077 factor B1 and GE23077 factor B2, a mixture of said factors in any proportion, the pharmaceutically acceptable salts and compositions thereof, and their use as an antibacterial agent with a selective inhibitory activity against E. coli RNA polymerase.
Another object of the present invention is a process for preparing GE23077 complex, namely GE23077 factor A1, GE23077 factor A2, GE23077 factor B1 and GE23077 factor B2, a mixture of said factors in any proportion, hereinafter reported as GE23077 compounds.
Actinomadura sp. DSMZ 13491 was isolated from a soil sample and deposited on May 22, 2000, with the DSMZ, (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-38124 Braunschweig, Germany), under the provision of the Budapest Treaty. The strain was accorded accession number DSMZ 13491.
The production of compounds GE23077 factors A1, A2, B1 and B2, is achieved by cultivating an Actinomadura strain capable of producing them, i.e. Actinomadura sp. DSMZ 13491 or a variant or mutant thereof; isolating the resulting antibiotic from the mycelium and or the culture broth; purifying the isolated antibiotic; and separating the antibiotic four factors A1, A2, B1 and B2 by chromatographic means. Wishing to produce the GE23077 complex, the separating step is evidently unrequired. In any case, it is preferred to produce compounds GE23077 under aerobic conditions in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen, and inorganic salts, purifying the resulting compounds by means of a chromatographic technique. Many of the nutrient media usually employed in the fermentation field can be used, however certain media are preferred.
Preferred carbon sources are glucose, xilose, cellobiose, cellulose, starch, corn meal, and the like. Preferred nitrogen sources are ammonia, nitrates, soybean meal, peptone, meat extract, yeast extract, tryptone, aminoacids, hydrolized casein and the like. Among the inorganic salts which can be incorporated in the culture media, there are the customary soluble salts capable of yielding sodium, potassium, iron, zinc, cobalt, magnesium, calcium, ammonium, chloride, carbonate, sulphate, phosphate, nitrate, and the like ions.
Preferably, the strain producing compounds GE23077 is pre-cultured in a fermentation tube or in a shake flask, then the culture is used to inoculate jar fermentors for the production of substantial quantities of substances. The medium used for the pre-culture can be the same as that employed for larger fermentations, but other media can also be employed. The strain producing compounds GE23077 can be grown at temperature between 20xc2x0 C. and 40xc2x0 C., preferably between 28xc2x0 C. and 37xc2x0 C.
During the fermentation, the GE23077 factors can be monitored by bioassay on susceptible microorganisms and/or by testing treated broth samples against or E. coli RNA polymerase and/or by HPLC analyses. Maximum production of GE23077 compounds generally occurs between the fourth and the seventh day of fermentation.
Compounds GE 23077 are produced by cultivating Actinomadura sp. DSMZ 13491 or a variant or mutant thereof producing compounds GE23077, and are found in the culture broths and/or in the mycelium.
Actinomadura sp. DSMZ 13491 grows well on many standard solid media. Microscopic examination and cell dimensions were measured using the culture grown on one-tenth strength humic acid medium (H. Nonomura, 1984xe2x80x94Design of a new medium for isolation of soil actinomycetes. The Actinomycetes 18, 206-209).
After seven days incubation at 28xc2x0 C., the strain revealed extensively branched vegetative hyphae (0.5 xcexcm in diameter). No fragmentation was observed. The aerial mycelium contained slightly twisted chains of large spores (1.2-1.4 xcexcm in diameter). No pseudosporangia were observed. Spore dimensions exceeded those of the mycelium, giving rise to a xe2x80x9csegmentedxe2x80x9d appearance of the spore chain.
Actinomadura sp. DSMZ 13491 was grown for seven days in AF-MS liquid medium (see Example 1 for medium composition). The mycelium was harvested by centrifugation and washed twice in sterile one quarter strength Ringer""s solution (OXOID). Subsequently, sufficient Ringer""s solution was added to the mycelium to provide a suitable inoculum. Aliquots of the suspension were streaked in a cross-hatched manner onto various media recommended by Shirling and Gottlieb (E. B. Shirling and D. Gottlieb, 1966xe2x80x94Method for Characterization of Streptomyces speciesxe2x80x94Int. J. Syst. Bacteriol., 16, 313-340) and several media recommended by Waksman (Waksman S. A., 1961xe2x80x94The Actinomycetesxe2x80x94The Williams and Wilkins Co., Baltimore. Vol 2, pp 328-334).
The ability to use a variety of carbohydrates as a carbon and energy source was determined in ISP8 medium (Shirling and Gottlieb, ibid) containing the carbon source at a final concentration of 2% (w/v). All media were incubated at 28xc2x0 C. for 21 days. Colour was assessed in natural daylight, using the Colour Atlas of Maerz and Paul (A. Maerz and M. R. Paul, 1950xe2x80x94A Dictionary of Colour, 2nd edition. McGraw-Hill Book Co. Inc., New York).
Colonial appearance, substrate and aerial mycelium colour and pigment production for strain GE23077 are recorded in Table I. Physiological characteristics of the strain are presented in Table II. The ability to utilise various carbohydrates for growth is shown in Table III.
Actinomadura sp. DSMZ 13491 was grown in Sauton""s medium for four weeks and the mycelium harvested, washed three times with sterile distilled water and subsequently freeze-dried. The stereoisomeric form of the diaminopimelic acid (DAP) was determined according to the method of Staneck and Roberts, (J. L. Staneck and G. D. Roberts, Simplified approach to identification of aerobic actinomycetes by thin-layer chromatography, Appl. Microbiol. 28, 226-231, 1974).
The whole cell sugar pattern was determined according to Saddler et al. (Saddler G. S., P. Tavecchia, S. Lociuro, M. Zanol, L Colombo and E. Selva. Analysis of madurose and other actinomycete whole cell sugars by gas chromatography. J. Microbiol. Meth., 14, 185-191, 1991).
Isoprenoid quinones were extracted and purified using the small scale integrated procedure of Minnikin et al. (D. E. Minnikin, A. G. O""Donnell, M. Goodfellow., G. Alderson, M. ALhalye, A. Schaal and J. H. Parlett. An integrated procedure of isoprenoid quinones and polar lipids. J. Microbiol. Meth. 2, 233-241, 1984).
The menaquinones were separated by HPLC and identified by their retention behaviour according to their isoprenylic chain length and degree of saturation, as described by Kroppenstedt (R. M. Kroppenstedt, Separation of bacterial menaquinones by HPLC using reverse phase RP18 and a silver loaded ion exchanger as stationary phase. J. Liquid. Chromat. 5: 2359-2367, 1982).
Polar lipids were extracted, examined by two dimensional thin layer chromatography and identified using published procedures (D. E. Minnikin, A. G. O""Donnell, M. Goodfellow, G. Alderson, M. Athalye, H. Schaal and J. H. Parlett. An integrated procedure of isoprenoid quinones and polar lipids. J. Microbiol. Meth. 2, 233-241, 1984).
For the extraction of fatty acids, the wet biomass was extracted using minor modifications (L. D. Kuykendall, M. A. Roy, J. J. O""Neill and T. E. Devine, Fatty acid, antibiotic resistance, and deoxyribonucleic acid homology groups of Bradyrhizobium japonicium, Int. J. System. Bact. 38, 351-361, 1988) of the method of Miller (L. T. Miller, A single derivatization method for bacterial fatty acid methyl esters including hydroxy acids, J. Clin. Microbiol. 16, 584-586, 1982). Analysis were carried out as described by Kroppenstedt (R. M. Kroppenstedt, E. Stackebrandt and M. Goodfellow, Taxonomic revision of the actinomycete genera Actinomadura and Microtetraspora, System. Appl. Microbiol. 13, 148-160, 1990) and data examined using the Microbial Identification System (L. T. Miller, ibid).
The strain DSMZ 13491 contains meso-2,6-diaminopimelic acid. Madurose is present in the whole-cell hydrolysate. As shown in FIG. 1, the more abundant menaquinone is MK-9 (H6) followed by smaller portions of MK-9(H4) and MK-9(H8). Among polar lipids, phosphatydilinositol, phosphatidylinositolmanosides, phosphatydilglycerol and diphosphatydilglycerol are identified in the chloroform methanol extracts. The following branched, saturated and unsaturated fatty acids plus tuberculostearic acid were detected.
Iso-15/17 Anteiso-15/17 Iso-16 10-Me16 10-Me17 10-Me18 2-OH xe2x88x92/xe2x88x92 xe2x88x92/xe2x88x92 + (+) + ++ xe2x88x92
(+): 1-5%; +: 5-15%; ++: 15-30%;
Iso-16: iso-hexadecanoic acid or 14-methylpentadecanoic acid;
10-Me-18: tuberculostearic acid;
2-OH-16: 2-hydroxy-palmitic acid.
The strain producing compounds GE23077 is assigned to the genus Actinomadura because of the following morphological and chemical characteristics:
the formation of a branched not fragmented vegetative myceliurn and of short chains of arthrospores;
the presence of meso-2,6-diamincpimelic acid in the cell wall and of madurose in the whole cell hydrolizate. This is characteristic of Chemotype IIIB according to Lechevalier and Lechevalier (H. A. Lechevalier and M. P. Lechevalier, A critical evaluation of the genera of aerobic actinomycetes, pp. 393-405; in: The Actinomycetales, H. Prausers ed., Jena, Gustav Fischer Verlag 1970);
the composition of polar lipids according to the Phospholipid type 1 sensu Lechevalier et al. (H. A. Lechevalier, C. De Brieve and M. P. Lechevalier, Chemotaxonomy of aerobic actinomycetes: phospholipid composition, Biochem. Syst. Ecol. 5: 246-260, 1977) and of Menaquinones type 4B2 according to Kroppensdedt (R. M. Kroppenstedt, Separation of bacterial menaquinones by HPLC using reverse phase RP18 and a silver loaded ion exchanger as stationary phase, J. Liquid Chromat. 5: 2359-2367, 1982; R. M. Kroppenstedt, E. Stackebrandt and M. Goodfellow, Taxonomic revision of the actinomycete genera Actinomadura and Microtetraspora, System. Appl. Microbiol. 13, 148-160, 1990);
the fatty acid profile of 3a Type according to Kroppenstedt and Goodfellow (R. M. Kroppenstedt and M. Goodfellow; The family Thermomonosporaceae, pp.1085-1114, in: The Prokariotes, Vol II, A. Balows, H. Truper, M. Dworkin, W. Harder and K. H. Schleifer eds; New York, Springer-Verlag, 1991).
As with other microorganisms, the characteristics of strain producing compounds GE23077 are subject to variation. For example, artificial variants and mutants of the strain can be obtained by treatment with various known mutagens, such as U.V. rays, and chemicals such as nitrous acid, N-methyl-Nxe2x80x2-nitro-N-nitrosoguanidine, and many others. All natural and artificial variants and mutants of strain Actinomadura sp. DSMZ 13491 are deemed equivalent to it for the purpose of this invention and therefore within the scope of invention.
The antibiotic may be recovered from the fermented broth, both from the mycelium and the supernatant fraction.
The recovery of GE23077 complex from the fermentation broths of the producing microorganism is conducted according to known per se techniques such as extraction with solvents, precipitation by adding non-solvents or by changing the pH of the solution, partition chromatography, reverse-phase partition chromatography, ion-exchange chromatography, molecular exclusion chromatography and the like.
A procedure for recovering the antibiotic substance of the invention from the fermentation broth includes extraction of GE23077 complex or the salts thereof, with water-immiscible organic solvents, followed by precipitation from the concentrated extracts, possibly by adding a precipitating agent.
The term xe2x80x9cwater-immiscible solventxe2x80x9d as used in this application, is intended to have the meaning currently given to it in the art and refers to solvents that, at the conditions of use, are slightly miscible or practically immiscible with water in a reasonably wide concentration range, suitable for the intended use.
Examples of water-immiscible organic solvents that can be used in the extraction of the compounds of the invention from the fermentation broths are: alkanols of at least four carbon atoms which may be linear, branched or cyclic such as n-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 1-hexanol, 2-hexanol, 3-hexanol, 3,3-dimethyl-1-butanol, 4-methyl-1-pentanol, 3-methyl-1-pentanol, 2,2-dimethyl-3-pentanol, 2,4-dimethyl-3-pentanol, 4,4-dimethyl-2-pentanol, 5-methyl-2-hexanol, 1-heptanol, 2-heptanol, 5-methyl-1-hexanol, 2-ethyl-1-hexanol, 2-methyl-3-hexanol, 1-octanol, 2-octanol, cyclopentanol, 2-cyclopentylethanol, 3-cyclopenthyl-1-propanol, cyclohexanol, cycloheptanol, cyclooctanol, 2,3-dimethylcyclohexanol, 4-ethylcyclohexanol, cyclooctylmethanol, 6-methyl-5-hepten-2-ol, 1-nonanol, 2-nonanol, 1-decanol, 2-decanol, and 3-decanol; ketones of at least five carbon atoms such as methylisopropylketone, methylisobutylketone, methyl-n-amylketone, methylisoamylketone and mixtures thereof.
As known in the art, product extraction may be mproved by adjusting the pH at an appropriate value, and/or by salting and/or by adding a proper organic salt forming an ion pair with the antibiotic which is soluble in the extraction solvent. As known in the art, phase separation may be improved by salting. When, following an extraction, an organic phase is recovered containing a substantial amount of water, it may be convenient to azeotropically distill water from it. Generally, this requires adding a solvent capable of forming minimum azeotropic mixtures with water, followed by the addition of a precipitating agent to precipitate the desired product, if necessary. Representative examples of organic solvents capable of forming minimum azeotropic mixtures with water are: n-butanol, benzene, toluene, butyl ether, carbon tetrachloride, chloroform, cyclohexane, 2,5-dimethyl furan, hexane, and m-xylene; the preferred solvent being n-butanol. Examples of precipitating agents are petroleum ether, lower alkyl ethers, such as ethyl ether, propyl ether, and butyl ether, and lower alkyl ketones such as acetone.
According to a preferred procedure for recovering of GE23077 complex, the filtered fermentation broths can be contacted with an adsorption matrix followed by elution with a polar water miscible solvent or a mixture thereof, concentration to water residue under reduced pressure, extraction with water-immiscible solvents, and precipitation with a precipitating agent of the type already mentioned above.
Examples of adsorption matrixes that can be conveniently used in the recovery of the compounds of the invention, are polystyrene or mixed polystyrene-divinylbenzene resins (e.g. M112 or S112, Dow Chemical Co.; Amberlite XAD2 or XAD4, Rohm and Haas; Diaion HP 20, Mitsubishi Chemicals), acrylic resins (e.g. XAD7 or XAD8, Rohm and Haas), polyamide resins such as polycaprolactames, nylons and cross-linked polyvinylpyrrolidones (e.g. Polyamide-CC 6, Polyamide-SC 6, Polyamide-CC 6.6, Polyamide-CC 6AC and Polyamide-SC 6AC, Macherey-Nagel and Co., west Germany; PA 400, M. Woelm AG, West Germany; and the polyvinylpirrolidone resin PVP-CL, Aldrich Chemie GmbH and Co., KG, West Germany), controlled pore cross-linked dextrans (e.g. Sephadex LH-20, Pharmacia Fine Chemicals, AB), and charcoal.
Preferably, polystyrene resins are employed, particularly preferred being the S112 (Dow Chemical Co.).
The preferred solvent for eluting GE23077 complex from the adsorption matrix depends on the specific stationary phase. In the case of polystyrene resins, polystyrene-divinylbenzene resins, acrylic resins or polyamide resin a preferred eluent is a water miscible solvent or its aqueous mixtures; in the case of charcoal a preferred eluent is a lower ketone such as acetone or a lower alcohol such as methanol. The aqueous mixtures can contain buffers at appropriate pH value.
The term xe2x80x9cwater-miscible solventxe2x80x9d as used in this application, is intended to have the meaning currently given in the art of this term and refers to solvents that, at the conditions of use, are miscible with water in a reasonably wide concentration range. Examples of water-miscible organic solvents that can be used in the elution of the compounds of the invention are: lower alkanols, e.g. (C1-C3) alkanols such as methanol, ethanol, and propanol; phenyl (C1-C3) alkanols such as benzyl alcohol; lower ketones, e.g. (C3-C4) ketones such as acetone and ethyl methyl ketone; cyclic ethers such as dioxane and tetrahydrofuran; glycols and their products of partial etherification such as ethylene glycol, propylene glycol, and ethylene glycol monomethyl ether, lower amides such as dimethylformamide and diethylformamide; acetic acid, dimethylsulfoxide and acetonitrile.
Purification of the crude GE23077 compounds, can be accomplished by any of the known per se techniques but is preferably conducted by means of chromatographic procedures.
Examples of these chromatographic procedures are those reported in relation to the recovery step and include also chromatography on stationary phases such as silica gel, alumina, activated magnesium silicate, and the like, with an organic eluting phase made of organic solvents including halogenated hydrocarbons, lower alkanols, ethers, higher ketones and mixtures thereof, or reverse phase chromatography on silanized silica gel having various functional derivatizations and eluting with an aqueous mixture of water-miscible solvents of the kind mentioned above.
Another way of purification is the chromatography on ion-exchange resin column. The elution can be conducted by a variation of pH or ionic-strength.
Conveniently, also the so called steric exclusion chromatography technique can be employed with good purification results. In particular, controlled pore cross-linked dextrans wherein most hydroxyl groups are alkylated, e.g. Sephadex LH-20 (Pharmacia Fine Chemicals, AB), are usefully employed in this technique.
For instance, medium pressure liquid chromatographic separation systems may be employed, using reverse phase chromatography on RP-8 or RP-18 functionalised silica gel and eluting with a sodium sulphate buffer.
As usual in this field, the production as well as the recovery and purification steps may be monitored by a variety of analytical procedures including HPLC and/or bioassay with a susceptible microorganisms and/or the inhibition assay of E. coli RNA polymerase.
The purification of individual factors A1, A2, B1 and B2 may be conveniently carried out by semipreparative HPLC of the GE23077 complex preparations.
A preferred preparative HPLC technique for the isolation of pure GE23077 factors A1, A2, B1 and B2 is performed on a semipreparative HPLC instrument (Shimadzu-LCBA) equipped with a 250xc3x9710 mm Supelcosil LC8 column, 5 xcexcm, (Supelco Inc; Bellefonte, USA), eluted at 4 ml/min flow rate with a 25 min linear gradient from 50% to 80% of phase B, followed by 5 min elution with 80% of phase B. Phase A is methanol: 100 mM ammonium sulphate pH 7 buffer 5:95 (v/v), and Phase B is methanol:water 2:8 (v/v). UV detection is at 230 nm. The eluates of repeated chromatographic runs containing the separated GE23077 factors are pooled according to their content and are concentrated under reduced pressure to aqueous solutions, which are freeze-dried yielding purified GE23077 factors A1, A2, B1 and B2.
As usual in this field, the production as well as the recovery and the purification steps may be monitored by a variety of analytical procedures including bioassay with susceptible microorganisms and/or inhibition tests on bacterial RNA polymerase, and/or TLC and/or HPLC procedures. A preferred analytical HPLC technique is performed on a HP 1090 instrument equipped with a 250xc3x974.6 mm column packed with C18 Ultrasphere ODS 5 xcexcm stationary phase (Beckmann Co.), eluted at 1 ml/min flow rate with mixture of phase A and B. Phase A was methanol:100 mM ammonium sulphate buffer 5:95 (v/v) and phase B was methanol:water 20:80 (v/v). Elution was carried out with a linear gradient from 50% to 80% of phase B in 20 min; 80% of phase B for 5 min. Typical retention times of the four GE23077 factors are: 14.4 (A1), 16.5 (B1), 19.4 (A2), 21.3 (B2).
The GE23077 complex is constituted of two couples of isomers: factors A1, A2 and factors B1, B2. It was observed that the individual pure Factor A1 and Factor A2, when kept in water solution or in mixtures of water miscible solvents and water solutions, reach an equilibrium state between them. The same behaviour was observed for pure Factor B1 and Factor B2. The equilibration rate was accelerated at acidic and basic pHs. Recording the NMR spectra of the GE 23077 factors A2 and B2 in DMSO, it has further been found that they fully converted (it took 12 hours for A2 and few minutes for B2, both at room temperature) into factors A1 and B1, respectively. Accordingly, if recorded after complete conversion, the spectra relating to factors A2 and B2 correspond to the ones of the factors A1 and B1, respectively.
Since compounds GE23077 complex and its factors contain acid functions, they are capable of forming salts with suitable bases according to conventional procedures. The antibiotics, when obtained in the acid form, may be converted into a corresponding non-toxic pharmaceutically acceptable salt. Suitable salts include the alkali and alkaline earth metal salts, typically the sodium, potassium, calcium and magnesium salts, and the ammonium and substituted ammonium salts. Representative substituted ammonium salts include primary, secondary or tertiary (CI-C4) alkylammonium and hydroxy (CI-C4) alkylammonium salts and, according to an embodiment of the present invention, the benzathine, procaine, hydrabamine and similar water insoluble, non-toxic, pharmaceutically acceptable salts. Also preferred, within said class of salts, are the salts of the compounds of the present invention commonly represented as the basic addition salts, i.e. the salts with basic aminoacids such as arginine or lysine, or aminosugars such as glucosamine and the like.
The alkali and alkaline earth metal salts are prepared according to the usual procedures commonly employed for preparing metal salts. As an example, antibiotic GE23077 is dissolved into the minimum amount of a suitable solvent, typically a lower alkanol, the stoichiometric amount of a suitable selected base is gradually added to the obtained solution and the obtained salt is precipitated by the addition of a non-solvent. The resulting alkali or alkaline earth metal salt is then recovered by filtration or evaporation of the solvents.
Alternatively, these salts can be prepared in a substantially anhydrous form by lyophilization; in this case, aqueous solutions containing the desired salts, resulting from the salification of antibiotic GE23077 with a suitably selected alkali or alkaline earth metal carbonate or hydroxide in such a quantity as to obtain a pH comprised between 7.0 and 8.5 are filtered from any insolubles and lyophilized.
The organic ammonium salts can be prepared substantially following the above procedure by adding the properly selected amine to a solution of antibiotic GE23077 in a suitable solvent and then evaporating off the solvent and the excess of the amine reagent or by lyophilizing the concentrate solution.
The pharmaceutically acceptable salts so formed are also part of this invention. xe2x80x9cPharmaceutically acceptablexe2x80x9d salts are salts which are useful in the therapy of warm-blooded animals.
The transformation of the compounds of the invention into the corresponding salts thereof, and viceversa, i.e. the transformation of a salt of a compound of the invention into the non-salt form are within the ordinary technical skill and are encompassed by the present invention.
A) Ultraviolet absorption spectrum, in a water:methanol 1:1 (v/v) solution, shows end-absorption (maximum at 204 nm), with no significative shift of wavelength absorbance at neutral and acidic pH. Upon addition of KOH, the maximum shifted at 218 nm. The spectrum was recorded on a Perkin-Elmer spectrophotometer mod. Lambda 16.
B) Positive ion FAB mass spectrometry analysis shows peaks corresponding to [Mxe2x88x92H]+ of the components of the complex and having 804 and 806 m/z. The FAB mass spectrometry analysis was carried out on a Finnigan TSQ700 triple quadrupole mass spectrometer using a xenon atom gun, operating at 8 kV, 0.23 mA current and glycerol as ionization matrix.
C) Amino acid analysis of the acid hydrolysate which shows the presence of valine, serine, threonine, isoserine, glycine and 2,3 diaminopropanoic acid, and other unidentified fragments.
The GE23077 complex was treated for 24 hours at 105xc2x0 C. with 500 xcexcl of 6N HCl in the presence of phenol, using a Pico-Tag apparatus (Millipore-Waters Co.). The residue was diluted with water and freeze-dried. The mixture was then treated sequentially a) at 100xc2x0 C. for 30 min with 200xcexcl of 2.4N HCl in n-butanol, dried and then treated at 100xc2x0 C. for 10 min with 100 ml of trifluoroacetic anhydride. The sample was then dried under nitrogen and dissolved in 100 ml of hexane, before submitting it to GC-MS analysis. The analysis was done using a Finnigan TSQ 700 triple stage GC/MS instrument with a SPB1 column, 30 mmxc3x970.2 mm (Supelco Inc; Bellefonte, USA) having a 0.25 xcexcm film thickness. The oven temperature was 60xc2x0 C. for 1 minute followed by a gradient from 60xc2x0 C. to 260xc2x0 C, at 12xc2x0 C./min; the carrier gas was helium at 8 Psi and split vent was at 80 ml/min; the injector temperature was 260xc2x0 C. The Electron Impact (EI) conditions were: EI positive ionization; source temperature: 150xc2x0 C.; electron energy: 70 eV and filament current: 400 ma. Chromatographic peaks were identified on the basis of their retention times and MS fragmentations.
D) Infrared absorption spectrum (shown in FIG. 2) which exhibits the following absorption maxima xcexd (cmxe2x88x921): 3292; 3072; 2955; 2924 (nujol); 2853 (nujol); 1732; 1686; 1655; 1628; 1545; 1462; 1377; 1317; 1263; 1219; 1113; 1049; 978; 721. The spectrum was recorded in nujol mull with an IFS-48 Fourier Transform spectrophotometer.
E) Retention times of the four GE23077 factors: 14.16 min (A1), 16.56 min (B1), 20.90 min (A2), 22.71 min (B2), which were found by HPLC analysis under the following chromatographic conditions (method A):
Instrument: HP mod. 1090 (DAD detector);
Column: Beckmann ODS C18 (5 xcexcm 250xc3x974.6 mm);
Elution: Isocratic 15% Phase B;
Phase A: Ammonium formiate (2.5 g/l): Methanol (99:1 v/v);
Phase B: Ammonium formiate (2.5 g/l): Methanol (30:70 v/v);
Flow rate: 1.5 ml/min;
Detector: UV 230 nm.
F) THe 1H-NMR spectrum (shown in FIG. 3), was recorded at 600 mHz in DMSO-d6.
9.00; 8.93; 6.34; 4.65; 4.47; 4.04; 3.99; 3.90; 3.88; 3.74; 3.53, 2.53; 1.95; 1.87; 1.71; 1.67; 0.96; 0.87; 0.85.
G) Rf value of 0.7 when analyzed by TLC using silica gel plates Merck 5714 (E. Merck; Darmstadt F. R. Germany) and developing in ethanol:n-butanol:water 2:2:1 (v/v). Detection was by scrubbing portions of the the silica layer, by extracting with methanol and by testing with the RNA polymerase assay the extracts.
A) positive ion FAB mass spectrometry analysis showed a peak corresponding to [Mxe2x88x92H]+ and had 804 m/z. The FAB mass spectrometry analysis was carried out on a Finnigan TSQ700 triple quadrupole mass spectrometer using a xenon atom gun, operating at 8 kV, 0.23 mA current and glycerol as ionization matrix.
B) 1H-NMR spectrum (shown in FIG. 4) was recorded at 600 MHz in DMSO-d6 (hexadeuterodimethylsulfoxide) and exibits the following signals (in ppm) referenced to the residual peak of DMSO set at 2.5 ppm as internal standard: the values marked with an asterisk are overlapping of two 1H signals.
8.94; 8.93; 8.93*; 8.04; 7.93; 7.88; 7.68; 7.33/7.27; 7.29; 6.34; 5.97; 5.79; 5.68; 5.00; 4.94; 4.82; 4.65; 4.47; 4.37; 4.31; 4.07; 4.05; 3.90; 3.88; 3.74; 3.59/3.49; 3.53,3.42/3.25; 2.50; 1.74; 1.70; 0.96; 0.94; 0.85.
C) 13C-NMR spectrum (shown in FIG. 5) was recorded at 150 MHz in DMSO-d6 (hexadeuterodimethylsulfoxide) and exibits the following signals (in ppm) referenced to the residual peak of DMSO set at 39.5 ppm as internal standard: the values marked with an asterisk are overlapping of two 13C signals.
175.6; 171.5; 171.3; 170.7; 170.2; 170.0; 170.0*; 169.6; 169.2; 168.8; 131.6; 129.9; 74.0; 69.9; 69.1; 66.8; 63.1; 60.1; 58.5; 58.2; 57.3; 55.9; 54.3; 43.4; 39.7; 29.8; 19.9; 19.3; 19.2; 13.7; 12.3.
D) Factor A1 analysed by the method A shows a retention time of 14.16 min.
A) positive ion FAB mass spectrometry analysis showed a peak corresponding to [Mxe2x88x92H]+ and had 804 m/z. The FAB mass spectrometry analysis was carried out on a Finnigan TSQ700 triple quadrupole mass spectrometer using a xenon atom gun, operating at 8 kV, 0.23 mA current and glycerol as ionization matrix.
B) HPLC analysis:
Factor A2 analyzed by the method A shows a retention time of 20.90 min.
C) 1H-NMR spectrum (recorded after complete conversion of factor A2 into factor A1, identical to the one shown in FIG. 4) was recorded at 600 MHz in DMSO-d6 (hexadeuterodimethylsulfoxide) and exibits the following signals (in ppm) referenced to the residual peak of DMSO set at 2.5 ppm as internal standard: the values marked with an asterisk are overlapping of two 1H signals.
8.94; 8.93; 8.93*; 8.04; 7.93; 7.88; 7.68; 7.33/7.27; 7.29; 6.34; 5.97; 5.79; 5.68; 5.00; 4.94; 4.82; 4.65; 4.47; 4.37; 4.31; 4.07; 4.05; 3.90; 3.88; 3.74; 3.59/3.49; 3.53,3.42/3.25; 2.50; 1.74; 1.70; 0.96; 0.94; 0.85.
D) 13C-NMR spectrum (recorded after complete conversion of factor A2 into factor A1, identical to the one shown in FIG. 5) was recorded at 150 MHz in DMSO-d6 (hexadeuterodimethylsulfoxide) and exibits the following signals (in ppm) referenced to the residual peak of DMSO set at 39.5 ppm as internal standard: the values marked with an asterisk are overlapping of two 13C signals.
175.6; 171.5; 171.3; 170.7; 170.2; 170.0; 170.0*; 169.6; 169.2; 168.8; 131.6; 129.9; 74.0; 69.9; 69.1; 66.8; 63.1; 60.1; 58.5; 58.2; 57.3; 55.9; 54.3; 43.4; 39.7; 29.8; 19.9; 19.3; 19.2; 13.7; 12.3.
A) positive ion FAB mass spectrometry analysis showed a peak corresponding to [Mxe2x88x92H]+ and had 806 m/z. The FAB mass spectrometry analysis was carried out on a Finnigan TSQ700 triple quadrupole mass spectrometer using a xenon atom gun, operating at 8 kV, 0.23 mA current and glycerol as ionization matrix.
B) The 1H-NMR spectrum (shown in FIG. 6) was recorded at 600 MHz in DMSO-d6 (hexadeuterodimethylsulfoxide) and exibits the following signals (in ppm) referenced to the residual peak of DMSO set at 2.5 ppm as internal standard: the values marked with an asterisk are overlapping of two 1H signals.
9.00; 8.99; 8.94; 8.02; 7.93; 7.88; 7.82; 7.32/7.26; 7.30; 5.90; 5.81; 5.66; 4.99; 4.96; 4.79; 4.65; 4.47; 4.31; 4.31; 4.07; 4.05; 3.88; 3.88*; 3.74; 3.57/3.37; 3.53; 3.47/3.23; 2.49; 1.95; 1.95*; 0.95; 0.94; 0.85; 0.84.
C) The 13C-NMR spectrum (shown in FIG. 7) was recorded at 150 MHz in DMSO-d6 (hexadeuterodimethylsulfoxide) and exibits the following signals (in ppm) referenced to the residual peak of DMSO set at 39.5 ppm as internal standard: the values marked with an asterisk are overlapping of two 13C signals.
175.5; 172.3; 171.4; 171.3; 170.7; 170.2; 169.9; 169.9*; 169.5; 169.0; 73.9; 70.0; 69.0; 66.8; 63.1; 60.0; 58.4; 58.3; 57.3; 55.9; 54.5; 44.5; 43.5; 39.4; 29.8; 25.4; 22.3; 19.9; 19.2; 19.2.
D) HPLC analysis:
Factor B1 analysed by method A shows a retention time of 16.56 min.
A) positive ion FAB mass spectrometry analysis showed a peak corresponding to [Mxe2x88x92H]+ and had 806 m/z. The FAB mass spectrometry analysis was carried out on a Finnigan TSQ700 triple quadrupole mass spectrometer using a xenon atom gun, operating at 8 kV, 0.23 mA current and glycerol as ionization matrix.
B) Factor B2 analysed by method A shows a retention time of 22.71 min.
C) The 1H-NMR spectrum (recorded after complete conversion of factor B2 into factor B1, identical to the one shown in FIG. 6) was recorded at 600 MHz in DMSO-d6 (hexadeuterodimethylsulfoxide) and exibits the following signals (in ppm) referenced to the residual peak of DMSO set at 2.5 ppm as internal standard: the values marked with an asterisk are overlapping of two 1H signals.
9.00; 8.99; 8.94; 8.02; 7.93; 7.88; 7.82; 7.32/7.26; 7.30; 5.90; 5.81; 5.66; 4.99; 4.96; 4.79; 4.65; 4.47; 4.31; 4.31; 4.07; 4.05; 3.88; 3.88*; 3.74; 3.57/3.37; 3.53; 3.47/3.23; 2.49; 1.95; 1.95*; 0.95; 0.94; 0.85; 0.84.
D) The 13C-NMR spectrum (recorded after complete conversion of factor B2 into factor B1, identical to the one shown in FIG. 7) was recorded at 150 MHz in DMSO-d6 (hexadeuterodimethylsulfoxide) and exibits the following signals (in ppm) referenced to the residual peak of DMSO set at 39.5 ppm as internal standard: the values marked with an asterisk are overlapping of two 13C signals.
175.5; 172.3; 171.4; 171.3; 170.7; 170.2; 169.9; 169.9*; 169.5; 169.0; 73.9; 70.0; 69.0; 66.8; 63.1; 60.0; 58.4; 58.3; 57.3; 55.9; 54.5; 44.5; 43.5; 39.4; 29.8; 25.4; 22.3; 19.9; 19.2; 19.2.
On the basis of she physico-chemical data reported above, the following structure formula can be tentatively assigned to antibiotic GE23077 complex, which is a preferred embodiment of the invention together with the pharmaceutically acceptable salts thereof: 
wherein
R is 
xe2x80x83for factors A1 and A2
and R is 
xe2x80x83for factors B1 and B2 and the pharmaceutically acceptable salts thereof.
The inhibition of RNA polymerase was determined in a cell free transcription assay performed in standard U-bottom 96-well plates. The [H3]-UTP incorporation in RNA was measured in the material precipitated upon addition of trichloroacetic acid (TCA). The reaction mixture contained 50 mM Tris-HCl (pH8), 50 mM KCl, 10 mM MgCl2, 0.1 mM EDTA, 5 mM dithiothreitol (DTT), 10 xcexcg/ml BSA, 20: g/ml calf thymus DNA, 1 mM ATP, 1 mM CTP, 1 mM GTP, 0.5 mCi 3H-UTP and 0.5 U of E. coli RNA polymerase enzyme (Epicentre Technology; Madison Wis.). 5 xcexcl of the tested solution were added to 45 xcexcl of reaction mixture, incubated at 37xc2x0 C. for 15 minutes and then quenched with 150 xcexcl of ice-cold 10% (w/v) TCA. After 30 min in ice, the well content was collected on glass-fiber filters (Filtermat A, Wallac) using a 96 wells cell harvester (Wallac) and radioactivity was determined in a xcex2-Plate scintillation counter (Wallac). Count per min (CPM) values are transformed in % of RNA polymerase inhibition by using the following formula:
% of RNA polymerase=100xe2x88x92[(CPM compoundxe2x88x92CPM blank)/(CPM controlxe2x88x92CPM blank)]*100
where: CPM compound is CPM in well with compound; CPM blank is CPM average in wells without enzyme template and CPM control is CPM average in wells without compound.
The GE23077 complex showed IC50 of E. coil RNA polymerase at 0.02 xcexcg/ml. A rifampicin resistant RNA polymerase (Promega; Madison Wis.) was inhibited with IC50=0.04 xcexcg/ml. Wheat germ RNA polymerase (Epicentre Technology; Madison Wis.) was inhibited at higher concentration (IC50=100 pg/ml).
The individual GE23077 factors inhibited the E. coli RNA polymerase, showing IC50=0.15 xcexcg/ml (Factor A1); 0.035 xcexcg/ml (Factor A2); 0.1 xcexcg/ml (Factor B1) and 0.02 xcexcg/ml (Factor B2).
Antimicrobial activity of complex GE23077 was determined using microdilution method with standard U-bottom 96-well plates according to The National Committee for Clinical Laboratory Standards; Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically- Third Edition; Approved Standard. NCCLS document M7-A3 Vol.13 No. 25.
The media used were cation-adjusted Mueller Hinton Broth (CAMHB) for Escherichia coli, Staphylococcus aureus, Moraxella catarrhalis, Bacillus subtilis, Mycobacterium smegmatis; Todd Hewitt Broth (THB) for Streptococcus pyogenes; Brain Heart Infusion Broth+1% (v/v) supplement C (CBHI) for Haemophilus influenzae; GC base Broth+1% (v/v) Isovitalex for Neisseria gonorrhoeae; Tripticase Soy Broth+10% fetal calf serum (TB) for Corynebacterrium jeikelum; Terrific Broth (TB) buffered with 50 mM Sodium Phosphate (pH 7) for E. coli sp. and E. coli ATCC 25922. Unless otherwise indicated inocula were 104 CFU/ml. All strains were incubated at 35xc2x0 C. in air, except H. influenzae and N. gonorrhoeae which were incubated in 5% CO2. Incubation time was 18-24 hours except for Moraxella catarrhalis, Neisseria gonorrhoeae, Haemophilus infiluenzae and Mycobacterium smegmatis that were grown for 48 hours. Visual readings were performed after incubation and the MIC was defined as the lower concentration that completely inhibited growth of tested microorganisms.
GE23077 complex is not active against most of the bacteria tested although it inhibits the growth of three strains of M. catarrhalis, with MIC in the 4-8 xcexcg/ml range. These strains are clinical isolates and are reported to have different levels of susceptibility to xcex2-lactams, as reported below.
GE23077 complex shows also marginal activity (MIC 256 xcexcg/ml) against N. gonorrhoeae ISM68/126, clinical isolate.
The individual factors A1, A2, B1 and B2 also inhibit M. catarrhalis. This was demonstrated by collecting the eluates of an HPLC fractionation of the GE23077 complex, by concentrating them under vacuum and by testing their activity against M. catarrhalis. The fractions containing the separated factors A1, A2,B1 and B2 inhibited the test microorganism.
Compounds GE23077 are thus inhibitors of M. catarrhalis. 
M. catarrhalis is a recognized important pathogen of humans. It is a common cause of respiratory tract infections, particularly otitis media in children and lower respiratory tract infections in the eldery. The widespread production of xcex2-lactamase enzyme renders M. catarrhalis resistant to the penicillins (K. McGregor, B. J. Chang, B. J. Mee and T. V. Riley. Moraxella catarrhalis: clinical significance, antimicrobial susceptibility and BRO beta-lactamases. Eur. J. Microbiol. Infect. Dis. 17, 219-34, 1998). M. catarrhalis has been recently accepted as the third commonest pathogen of the respiratory tract after Streptococcus pneumoniae and Haemophilus influenzae (M. C. Enright and H. McKenzy, Moraxella (Branhamella) catarrhalisxe2x80x94Clinical and molecular aspect of a rediscovered pathogen, J. Med. Microbiol. 46, 360-71, 1997).
The compounds of the invention can be administered, as a pharmaceutically acceptable composition, as such or in admixture with a pharmaceutically acceptable carrier and can also be administered in conjunction with other antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and glycopeptides. Conjunctive therapy thus includes sequential, simultaneous and separate administration of the active compound in a way that the therapeutic effects of the first administered one is not entirely disappeared when the subsequent is administered.
The compounds of the invention can be accordingly used as a medicament; the single factors A1, A2, B1 and B2 can be utilized alone or as a mixture of two or more of them, in any proportion. Said mixture may be obtained by mixing predetermined amounts of two or more factors. Alternatively, mixtures of the four factors can be directly obtained from the isolation of the fermentation product of Actinomadura sp. DSMZ 13491 according to the above described process. An example of said mixture is the C-E23077 complex which is constituted by the factors A1, A2, B1 and B2.
Preferably, the compounds of the invention, are formulated into formulations suitable for parenteral administration, according to procedures known per se in the art and reported in reference books such as the one mentioned above.
For instance, a compound of the invention is formulated with a solubilising agent, such as polypropylene gliycol or dimethylacetamide, and a surface-active agent, such as polyoxyethylene sorbitan mono-oleate or polyethoxylated castor oil in sterile water for injection.
An example of a typical formulation for parenteral administration contains 10 mg of antibiotic G72307, factors per ml of final preparation, 10-20% of a surface-active agent, which may be a polyoxyethylene sorbitan fatty acid ester, a polyoxyethylene castor oil derivative or a polyoxyethylene hydrogenated castor oil derivative and 0-20%, preferably 10-20%, of a solubilizing agent such as propylene glycol, dimethylacetamide, dimethylformamide, ter-butyl-N-hydroxycarmabate, 1,2-, 1,3-, or 1,4-butandiol, ethyl oleate, tetrahydrofurfuryl-polyethylene-glycol 200, dimethyl isosorbide, benzyl alcohol and the like. A preferred solubilizing agent is propylene glycol.
Polyoxyethylene sorbitan fatty acid esters are commercially available and some of them are traded under the trade name xe2x80x9cTweenxe2x80x9d. They are also known with the non-proprietary name of xe2x80x9cpolysorbatesxe2x80x9d. Examples of them are polysorbate 20, 21, 40, 60, 61, 65, 80, 81 and 85. Preferred for use in the formulaticns of the invention is polysorbate 80 [sorbitan mono-9-octadecanoate, poly-(oxy-1,2-ethanediyl)derivatives].
Polyoxyethylene castor oils and polyoxyethylene hydrogenated castor oils are also commercially available. Some of them are traded with the trade name xe2x80x9cCremophorxe2x80x9d. Examples of such compounds are those known as Cremophor EL (polyethoxylated castor oil), Cremophor RH 40 (polyethoxylated hydrogenated castor oil), Cremophor RH 60 (PEG 60 hydrogenated castor oil) or Emophor EL-719 (polyoxyethylated vegetable oil).
Preferably, a formulation for injection should have a pH in the range of 7xc2x10.5. If necessary, it might be advisable to adjust the pH of the preparation with a suitable buffering agent. Conveniently, TRIS (i.e. trihydroxymethylaminomethane) or phosphate can be used as buffering agents. A preferred formulation fox parenteral administration includes the following excipients: propylene glycol from 5 to 20%, preferably 10-20%. Generally, these formulations can be preparec by dissolving the active ingredient into the organic solvent, then adding the surface active ingredient, and finally diluting to the desired volume with sterile water for injection.
Alternatively, the active ingredient may be prepared as a lyophilized powder for reconstitution before use.
If the lyophilized material is prepared starting from a mixture containing the active ingredient and the surfactant, such as polyethylene glycol 60 hydrogenated castor oil, it can conveniently be reconstituted with the aqueous medium alone, without addition of an organic solvent.
Optionally, a common lyophilization aid can be added, if necessary, to obtain a lyophilized material in powder form. Preferably, all these formulations are used for i.v. administration in the treatment of any infection involving a microorganism susceptible to the antibiotic of the invention.
Alternatively, the active ingredient may be prepared as a lyophilized powder for reconstitution before use. If the lyophilized material is prepared starting from a mixture containing the active ingredient and the surfactant, such as polyethylene glycol 60 hydrogenated castor oil, it can conveniently be reconstituted with the aqueous medium alone, without addition of an organic solvent. Optionally, a common lyophilization aid can be added, if necessary, to obtain a lyophilized material in powder form.
Preferably, all these formulations are used for i.v. administration in the treatment of any infection involving a microorganism susceptible to the antibiotic of the invention. The antibiotic may also be used in a suitable pharmaceutical form such as a capsule, a tablet or an aqueous suspension.
The dosage of the active ingredient depends on many factors which include type, age and conditions of the patient, specific active ingredient and formulation selected for the administration, administration schedule, etc. In general, effective antimicrobial dosages are employed per single unit dosage form. Repeated applications/administrations, e.g. from 2 to 6 times a day, are in general preferred. An effective dosage may be in general in the range 0.5-50 mg/kg body weight/day. A preferred topic preparation is an ointment containing from 1% to 10% of a compound of the present invention.
Anyway, the prescribing physician will be able to determine the optimal dosage for a given patient in a given situation.